Protein-Protein Variation of Protein Assays

Protein assays differ in their chemical basis for detecting protein-specific functional groups. Some assay methods detect peptide bonds, but no assay does this exclusively. Instead, each protein assay detects one or several different particular amino acids with greater sensitivity than than others. Consequently, proteins with different amino acid compositions produce color at different rates or intensities in any given protein assay.

The following table compares the protein-to-protein variability in color response of several Thermo Scientific Pierce Protein Assays. These data serve as a general guideline for evaluating response differences among protein samples. However, because the comparisons were made using one protein concentration and buffer, they should not be used as exact calibration factors.

This variability information is helpful for choosing a protein standard. For example, when the sample to be assayed is a purified antibody, bovine gamma globulin (BGG, protein #5) will be a more accurate standard than bovine serum albumin (BSA, protein #1). These data also indicate the importance of specifying which assay standard was used when reporting protein assay results.

For each of the protein assays presented here, 14 proteins were assayed using the standard test tube protocol. The net (blank corrected) average absorbance for each protein was calculated. The net absorbance for each protein is expressed as a ratio to the net absorbance for BSA (e.g., a ratio of 0.80 means that the protein produces 80% of the color obtained for an equivalent mass of BSA). All protein concentrations were at 1000µg/mL, except with the Micro BCA Assay which were at a concentration of 10µg/mL.

Results

BCA
(Note 1)

Micro
BCA

Modified
Lowry

Coomassie
Plus

Coomassie
(Bradford)

Pierce
660nm

Relative Uniformity

High

High

High

Medium

Low (Note 2)

Low

Coefficient of Variation

14.7%

11.4%

11.9%

28.8%

38.2%

37%

Standard Deviation

0.15

0.12

0.13

0.21

0.26

0.27

Average ratio

1.02

1.05

1.09

0.73

0.68

0.74

Tested Protein

^

^

^

^

^

^

1. Albumin, bovine serum

1.00

1.00

1.00

1.00

1.00

1.00

2. Aldolase, rabbit muscle

0.85

0.80

0.94

0.74

0.76

0.83

3. alpha-Chymotrypsinogen

1.14

0.99

1.17

0.52

0.48

—

4. Cytochrome C, horse heart

0.83

1.11

0.94

1.03

1.07

1.22

5. Gamma Globulin, bovine

1.11

0.95

1.14

0.58

0.56

0.51

6. IgG, bovine

1.21

1.12

1.29

0.63

0.58

—

7. IgG, human

1.09

1.03

1.13

0.66

0.63

0.57

8. IgG, mouse

1.18

1.23

1.20

0.62

0.59

0.48

9. IgG, rabbit

1.12

1.12

1.19

0.43

0.37

0.38

10. IgG, sheep

1.17

1.14

1.28

0.57

0.53

—

11. Insulin, bovine pancreas

1.08

1.22

1.12

0.67

0.60

0.81

12. Myoglobin, horse heart

0.74

0.92

0.90

1.15

1.19

1.18

13. Ovalbumin

0.93

1.08

1.02

0.68

0.32

0.54

14. Transferrin, human

0.89

0.98

0.92

0.90

0.84

0.8

15. a-Lactalbumin

—

—

—

—

—

0.82

16. Lysozyme

—

—

—

—

—

0.79

17. Trypsin inhibitor, soybean

—

—

—

—

—

0.38

Notes:
1. The BCA - Reducing Agent Compatible (BCA-RAC) Assay also produced a low coefficient of variation.
2. The Bio-Rad Bradford Protein Assay tested with the same proteins as our Coomassie (Bradford) Assay produced a very high coefficient of variation (46%), corresponding to very low relative uniformity.